Vaginal Health Products

ABSTRACT

The invention provides compositions and methods for increasing cell growth, stimulating cell turnover and promoting the secretion of mucus within the reproductive tract of a female mammal.

FIELD OF THE INVENTION

The present invention relates to the use of vitamin E and various planthormones for increasing the growth, renewal and mucus production ofvaginal epithelial cells and improving the health of the female genitaltract.

BACKGROUND OF THE INVENTION

As estrogen levels fall during menopause, estrogen dependent tissue willstart to involute and take on the characteristic appearance of estrogendeprivation. Cervical mucus levels diminish and vaginal mucosa regressesduring menopause. With aging, the vagina becomes shortened, ruggaedisappear, and elasticity is lost. Vaginal secretion becomes scanty.When estrogen is provided, some of these effects are reversed: thecervix may secrete more mucus and the vaginal mucosa may regain lostlayers. However, the symptoms often do not disappear completely, in partbecause the amount of estrogen provided for hormone replacement is lowerthan circulating estrogen levels during a normal menstrual cycle.

Approximately 40% of postmenopausal women experience atrophic vaginitisor vaginal dryness. During vaginal atrophy, the vaginal epitheliumdecreases in thickness, hydration, rugae (folds), and blood flow. Causesof atrophic vaginitis include a decrease in the amount of estrogenpresent both locally and systemically as well as environmental factorssuch as chemotherapy, antihistamines, smoking cigarettes, excessiveexercise, and perineal products (i.e. douches, deodorants, andperfumes). Estrogens or hormone replacement therapies (HRTs) areeffective in reducing vaginal dryness. However, possible dangerous sideeffects include a higher incidence of breast cancer, endometrial cancer,blood clots, nausea, breast tenderness, and headache.

Products that are available over-the-counter include lubricants such asAstroglide and KY Lubricating Jelly as well as moisturizers such asReplens and KY Long Lasting Moisturizer. These products, which aremostly water in composition, provide only temporary relief (1-2 days)for symptoms and provide virtually no long-term benefits to the vaginaltissue.

Therefore, vaginal dryness and regression of vaginal mucosa areproblematic, particularly after menopause. Stimulation of cervical mucusproduction can help alleviate vaginal dryness, and can also augment theaction of exogenously administered estrogen to alleviate vaginaldryness. Moreover compositions and methods for counteracting theregression of vaginal mucosa are also needed.

SUMMARY OF THE INVENTION

The invention provides non-hormonal therapies for treating certainreproductive and vaginal problems, including atrophic vaginitis. Ingeneral, these therapies have minimal side effects, stimulate naturaland non-hormonal mechanisms of action, promote vaginal cell growth andrenewal, increase mucus secretion, stimulate gene expression, replaceaging tissues with new tissues and maintain or restore healthy tissuefunction.

Thus, the present invention is directed to a variety of compositions andmethods for treating or preventing vaginal and/or reproductive problemsin a female mammal. The compositions involve an effective amount of acomposition that includes vitamin E and/or a plant hormone such asjasmonic acid or gibberellic acid. The compositions of the invention canalso include a retinoid or carotenoid (e.g. vitamin A) and/or one ormore nucleotide(s) or nucleoside(s). In general, these compositions canbe administered topically or intravaginally. The compositions andmethods of the invention can inter alia increase vaginal cell growth,stimulate renewal or turnover of vaginal cell layers, and/or toalleviate or diminish vaginal dryness in a female mammal. Thecompositions and methods of the invention can also stimulate theproduction of collagen and fibronectin.

In one embodiment, the invention provides a method to increase growth ofvaginal or cervical epithelial cells by administering to the femalemammal an effective amount of vitamin E and/or a plant hormone such asjasmonic acid or gibberellic acid. The compositions of the invention canalso include a retinoid or carotenoid (e.g. vitamin A) and/or one ormore nucleotide(s) or nucleoside(s).

In another embodiment, the invention provides a method to increaseexpression of mucin, for example, mucin-4, in vaginal or cervicalepithelial cells by administering to the female mammal a compositionhaving an effective amount of vitamin E and/or a plant hormone such asjasmonic acid or gibberellic acid. The compositions of the invention canalso include a retinoid or carotenoid (e.g. vitamin A) and/or one ormore nucleotide(s) or nucleoside(s).

In another embodiment, the method involves increasing the expression ofP2Y₂ receptors or estrogen receptors or vascular endothelial growthfactor in vaginal or cervical epithelial cells by administering to thefemale mammal a composition having an effective amount of vitamin Eand/or a plant hormone such as jasmonic acid or gibberellic acid. Thecompositions of the invention can also include a retinoid or carotenoid(e.g. vitamin A) and/or one or more nucleotide(s) or nucleoside(s).

Such methods can facilitate renewal of regressing vaginal mucosa,prevent or treat vaginal dryness in a mammal, or maintain or enhance thenormal protective function of vaginal mucus in a mammal. In general, thevitamin E, a plant hormone (e.g. jasmonic acid or gibberellic acid) andother compounds are administered intravaginally.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates that 1 μM vitamin E for 24 hours increases mucin-4expression. An ethidium stained 2.0% agarose gel is shown with separatedβ-actin (350 bp positive control) and mucin-4 (800 bp) PCR products.Lane 1 contains a DNA marker. Lanes 2-5 contain PCR products usingcervical cDNA, placenta cDNA, ME-180 cDNA (no treatment), and ME-180cDNA (1 μM vitamin E), respectively, as templates.

FIG. 2A-2C illustrates the effects of three concentrations oftrans-retinoic acid (circles), 9-cis-retinoic acid (square symbols), and13-cis-retinoic acid (diamonds) on ME-180 cell growth over a period oftwo days. As a negative control, the effect of media without retinoicacid (crosses) on cell growth is also illustrated. FIG. 2A graphicallyillustrates the effects of 10 μM trans-retinoic acid (circles),9-cis-retinoic acid (squares), and 13-cis-retinoic acid (diamonds) onME-180 cell growth over a period of two days. FIG. 2B graphicallyillustrates the effects of 1 μM trans-retinoic acid (circles),9-cis-retinoic acid (squares), and 13-cis-retinoic acid (diamonds) onME-180 cell growth over a period of two days. FIG. 2C graphicallyillustrates the effects of 100 nM trans-retinoic acid (circles),9-cis-retinoic acid (squares), and 13-cis-retinoic acid (diamonds) onME-180 cell growth over a period of two days. The number of ME-180 cellsis provided on the y-axes.

FIG. 3 provides a copy of photograph of an ethidium bromide-stained 2%agarose gel of PCR products using cDNA derived from ME-180 cervicalepithelial cell mRNA as template. Two sets of primers were used togenerate the products in lanes 2-11: P2Y₂-specific primers (top band,650 bp, lanes 2-11) and β-actin-specific primers (bottom band, 300 bp).Lanes 1 and 12 are DNA size markers. The P2Y₂ product can barely be seenin lanes 2, 3, and 7 where the template cDNA was from cervical cells(lane 2), ME-180 cells without vitamin A (lane 3), and ME-180 cellswithout vitamin A or serum (lane 7), respectively. The addition of 100nM vitamin A to ME-180 cells caused an increase in P2Y₂ expression inthe presence of serum (lanes 4-6) and in the absence of serum (lanes8-11).

FIG. 4 provides a copy of a photograph of an ethidium bromide-stained 2%agarose gel of PCR products using cDNA derived from ME-180 cervicalepithelial cells as template. Lane 1 provides DNA size markers. Two setsof primers were used to generate the PCR products in lanes 2-10:ER-α-specific primers (upper band) and β-actin-specific primers (lowerband). In Lane 2 the template was cDNA from ME-180 cells cultured in thepresence of serum. In Lanes 3 and 7, the template was cDNA from ME-180cells cultured in the absence of serum. In Lanes 4-6, the templates werecDNAs from ME-180 cells cultured without serum and treated with 100 nMvitamin A for 4, 8 and 16 hours, respectively. Addition of 100 nMvitamin A to ME-180 cells caused an increase in ER-α expression.

FIG. 5 provides a copy of a photograph of an ethidium bromide-stained 2%agarose gel of PCR products using RNA from various cell types astemplate. Lanes 1 and 7 provide DNA size markers. Two sets of primerswere used to generate the PCR products in lanes 2-6: vascularendothelial growth factor (VEGF) specific primers (upper band) andβ-actin-specific primers (lower band). Lanes 2 and 6 provide the PCRproducts from cDNA derived from untreated ME-180 cells. Lanes 3-5provide the PCR products from cDNA derived from ME-180 cells treatedwith 100 nM vitamin A for 4, 8, and 16 hours, respectively.

FIG. 6 provides a copy of a photograph of an ethidium bromide-stained 2%agarose gel of PCR products using RNA from various cell types astemplate. Lane 1 of the gel depicted in FIG. 6 contains DNA sizemarkers, in particular a 100 bp ladder of size markers (the brightestband is 600 bp). Lane 2 contains the products of a RT-PCR reaction usingtemplate cDNA derived from cervical RNA and mucin-4 primers (800 bpproduct) with β-actin primers (300 bp product). Lane 3 contains theproducts of a similar PCR reaction using template cDNA from ME-180cells. Lane 4 contains the products of a similar PCR reaction usingtemplate cDNA from ME-180 cells treated with 100 nM vitamin A for 24hours.

FIG. 7 graphically illustrates the effect of jasmonic acid andgibberellic acid on cell renewal time in healthy human volunteers, ascompared to human volunteers that received no jasmonic acid orgibberellic acid (control). As shown, cell renewal was slower involunteers that received no jasmonic acid and no gibberellic acid.Jasmonic acid increased the time required for cell renewal in human skinby about 28%, whereas gibberellic acid increased the cell renewal timeby about 20%.

FIG. 8 graphically illustrates the effect of jasmonic acid on cellgrowth over time as measured by hemocytometer counting. Control cells(open circles) receiving no jasmonic acid had the least cell growth.Cells receiving 1 μg/ml jasmonic acid (closed triangles), 10 μg/mljasmonic acid (X symbols), and 100 μg/ml jasmonic acid (closed diamonds)exhibited increased cell growth in a dose dependent manner.

FIG. 9 graphically illustrates the effect of jasmonic acid on cellgrowth over time as measured by direct cell counting. Control cells(open circles) receiving no jasmonic acid had the least cell growth.Cells receiving 1 μg/ml jasmonic acid (closed squares), 10 μg/mljasmonic acid (closed diamonds) and 100 μg/ml jasmonic acid (X symbols)exhibited increased cell growth in a dose dependent manner. These dataindicate that plant hormones such as jasmonic acid can increase cellgrowth in epidermal cells and may be useful in treating vaginal atrophy.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for increasing thesecretion of mucus, and stimulating cell growth and renewal of epidermallayers within the reproductive system of a female mammal. Thecompositions of the invention include vitamin E and/or a plant hormonesuch as jasmonic acid or gibberellic acid. Administration of suchcompositions can increase mucus production, stimulate cell growth andpromote the renewal of vaginal and cervical cells in the femalereproductive system. Thus, the compositions of the invention canfacilitate replacement of older cells with new cells, therebyrejuvenating the lining of the female reproductive tract.

This invention utilizes inexpensive, readily available active compoundsthat effectively enhance the natural ability of vaginal and cervicaltissues to renew themselves and to produce moisture. The methods andcompositions of the invention therefore avoid strong chemicals andunnatural substances whose effects on the health and reproduction of theuser are unknown.

The methods of the invention may improve the quantity and quality of thesecretions of the reproductive organs, repair and replace aging tissuesand influence expression of genes within reproductive, epithelial andmucosal cells. Genes whose expression may be influenced by the methodsof the invention include mucin genes.

Mucins refer to a family of glycoproteins of high molecular weight,secreted or expressed by goblet and nongoblet epithelial cells ofmucosal tissues. Mucins can form mucus, a highly hydrated gel ofparticular structure and function. Mucins from diverse species havesimilar structural features, particularly with regard to the mucinprotein backbone. Nine distinct mucin genes have been identified (MUC1,2, 3, 4, MUC5AC, MUC5B, MUC6, 7 and 8). Mucins are glycoproteinscontaining from fifty to eighty percent carbohydrate. They are large,elongated molecules (molecular weight 10⁵ to 10⁷ daltons) with a proteinbackbone to which oligosaccharides are attached in a bottle-brushconfiguration. The oligosaccharide side chains, or bristles, can behighly variable in their make-up, indicating that the more basicfunctions of the molecule derive from the protein core. These moleculescan be crosslinked through disulfide bridges to form very high molecularweight gels. Different tissues may produce different types of mucins.

According to the invention, vitamin E can increase the expression ofmucins and thereby provide increased secretion and formation of mucuswithin the reproductive tracts of female mammals.

Influences on mucus secretion that may be provided by the inventioninclude, but not limited to, the quantity and type of mucin (e.g. sulfoand/or sialomucin), changes in viscosity, hydrogen ion retardation,hydrophobicity, changes in phospholipid content, glycosylation andsulfation, macromolecular assembly, surface tension, adhesivity,transport properties, elastic modulus, tensile properties, rigidityfactors, recoil factors, spinnbarkeit, sperm penetration qualities,consistency, cellularity, ferning, and the like.

The methods of the invention can change the constitutive and stimulatedsecretions of the local reproductive system, including those of thevagina, cervix, uterus, fallopian tube, Bartholin or vestibular glandsand urethral secretions. The methods and compositions of the inventioncan influence the function of the mucus genes found in the reproductivesystem, including, but not limited to genes that control mucusproduction in the cervix, uterus, and Bartholin's glands and other partsof the reproductive system with mucus secreting cells. The squamousepithelium of the lower genital tract (vagina; for example) andepithelial cells of the cervix can be treated by the methods of theinvention. Included are methods to influence or change the secretaryeffects of the mucus genes, mucus secreting cells and cells thatinfluence the properties of secretory and cell surface mucins of all theabove mentioned glands of the reproductive system.

Mucus can be defined by its chemical, physical and biologicalproperties. Rheological or flow properties of mucus include viscosity,rate of flow, shear index, spinnbarkeit or stretch of mucus due toincreased viscoelasticity and ferning (crystallization) parameters.Changing or stimulating the hydration, viscosity, quantity or otherproperties of vaginal secretions can influence a variety of conditionsand disorders, including, but not limited to contraception, infertility,menopause, dyspareunia, infections, and others related and unrelatedconditions. Description of the function and anatomy of these organs canbe found in Novak's Gynecology, 12.sup.th edition, eds. Berek, Adashiand Hillard, Williams and Wilkins, Baltimore, Md., 1996.

The methods and compositions of the invention can also increase thegrowth of cells lining the female reproductive tract, for example,vaginal and cervical cells. Such increased cell growth may occur veryquickly, or after only a few days of treatment. For example, after onlytwo or three days of treatment the number of newly formed cells can bealmost twice or almost three times or four times or five times that ofuntreated individuals. As treatment progresses the number of newlyformed cells can increase further. For example, treated individuals mayhave about 2 to about 20 times the number of young, newly formed cellscompared to untreated individuals. Other individuals may have about 2 toabout 10 times the number of young, newly formed cells as untreatedindividuals. Such increased cell growth can repair and replace agingcells and tissue, rejuvenate the lining of the female reproductive tractand provide greater resilience and improved health to tissues involvedin reproduction.

The invention therefore has at least two general utilities. First, theinvention may increase the amount of mucus and/or the water content ofsecretions of the reproductive organs to improve the health and toincrease lubrication of the female reproductive system. Second, theinvention may be used to rejuvenate aging tissues and enhance the healthand resiliency of those tissues, for example, by stimulating cellulargrowth, gene expression and mucus secretion.

Vitamin E

There are at least eight naturally occurring compounds with vitamin Eactivity. Each vitamin E compound is a derivative of 6-chromanol. SeeULLMANN's ENCYCLOPEDIA OF INDUSTRIAL CHEMISTRY, Vol. A 27, Vitamin E,Chapter 4: 478-488 (VCH Verlagsgesellschaft, 1996). The tocopherol group(Compounds Ia-d) has a saturated side chain, while the tocotrienol group(Compounds IIa-d) has an unsaturated side chain. The tocopherol groupcomprises compounds of formula I:

wherein R₁, R₂ and R₃ are all separately hydrogen (H), methyl (CH₃) orhydroxyl (OH). The compound α-tocopherol (Ia) is a compound of formulaI, wherein R₁, R₂ and R₃ are all methyl. The compound β-tocopherol (Ib)is a compound of formula I, wherein R₁ and R₃ are methyl and R₂ ishydrogen. The compound γ-tocopherol (Ie) is a compound of formula I,wherein R₁ is hydrogen, while R₂ and R₃ are methyl. The compound6-tocopherol (Id) is a compound of formula I, wherein R₁ and R₂ arehydrogen, while R₃ is methyl.

The tocotrienol group comprises compounds of formula II:

wherein R₁, R₂ and R₃ are all separately hydrogen (H), methyl (CH₃) orhydroxyl (OH).

The compound α-tocotrienol (IIa) is a compound of formula II, whereinR₁, R₂ and R₃ are all methyl. The compound β-tocotrienol (IIb) is acompound of formula II, wherein R₁ and R₃ are methyl and R₂ is hydrogen.The compound γ-tocotrienol (IIc) is a compound of formula II, wherein R₁is hydrogen, while R₂ and R₃ are methyl. The compound 6-tocotrienol(IId) is a compound of formula II, wherein R₁ and R₂ are hydrogen, whileR₃ is methyl

Within the present invention, vitamin E is understood to include all ofthe above mentioned tocopherols and tocotrienols with vitamin Eactivity.

When present in the compositions of the present invention, vitamin Ecompounds can be used in an amount of about 0.0001% to about 50%, orfrom about 0.001% to about 20%, or from about 0.001% to about 10%, orfrom about 0.01% to about 5%, or from about 0.05% to about 2% of thecomposition. According to the invention, in situ concentrations of anactive gibberellic acid ranging from about 10⁻⁵ M to about 10⁻⁷ M areeffective for increasing epithelial cell proliferation and mucin-4expression.

Gibberellic Acid

Gibberellic acid comprises a class of compounds that is also referred toas gibberellins. Gibberellins are plant hormones that affect a widevariety of processes throughout the life cycle of plants, including seedgermination, stem elongation, flower induction, anther development, andseed and pericarp growth. Gibberellins are tetracyclic diterpenoid acidsthat can isolated from fungi and higher plants and that have theent-gibberellane ring system shown in the following structure (III).

Gibberellins were first isolated by Japanese researchers in the 1930sfrom cultures of the fungus Gibberella fujikuroi (Fusarium moniliforme).Gibberellins are secondary metabolites that have since been shown to bepresent in other fungal species, in some ferns, and in many gymnospermsand angiosperms. Of the 121 known gibberellins, 96 have been identifiedonly in higher plants, 12 are present only in Gibberella, and 12 arepresent in both. As observed in Gibberella, many different gibberellinscan be present in individual angiosperms.

Two main types of gibberellins exist: the C₂₀-gibberellins, which have20 carbon atoms (structure IV, below), and the C₁₉-gibberellins, inwhich the twentieth carbon atom has been lost due to metabolism(structure V, below). The carboxylic acid at carbon-19 bonds tocarbon-10 to produce a lactone bridge in almost all of theC₁₉-gibberellins.

The ent-gibberellane ring system can contain many structuralmodifications, accounting for the large number of known gibberellins.Naturally occurring gibberellins with identified structures areallocated an “A number” (MacMillan et al. (1968) Nature 217:170-171). Atpresent, 126 naturally occurring gibberellins of plant and fungal originare known. Current structural information on gibberellins can be foundat the website plant-hormones.bbsrc.ac.uk/gibberellin_information2.htm.

Variations in gibberellin structure arise in several ways. Carbon-20 canexist in different oxidative states, for example, methyl (—CH₃),hydroxymethyl (—CH₂OH), aldehyde (—CHO), or carboxylic acid (—COOH). Theent-gibberellane skeleton especially that of the C₁₉-gibberellins, canalso contain additional functional groups. Hydroxyl (—OH) groups arefrequently inserted into the ring system; insertion of epoxide (>O) andketone (═O) functions also occurs, although less commonly. The positionand/or stereochemistry of substituent groups can affect the biochemicaland physiological significance of the molecules. Substituent groupspositioned above the ring plane are said to be in the β-configuration;their bonding to the ring is designated by a solid, elongated triangle.Substituent groups positioned below the ring plane are said to be in theα-configuration; their bonding to the ring is designated by a dashed,elongated triangle. The attachment of substituent groups in the plane ofthe ring system is indicated by a straight line.

Gibberellins can exist as conjugates, for example, with a hexose orpentose molecule such as glucose. An ether or an ester linkage may linksuch a glucose molecule to the gibberellin. Such conjugation maytemporarily or permanently inactivate the activity of a gibberellinwithin a plant.

The biological activity of different gibberellins varies, and variousgibberellins within a plant can be precursors, biosyntheticintermediates, or deactivation products of active gibberellins. Threestructural features are commonly associated with gibberellin biologicalactivity: a 3-hydroxyl group, a 7-carboxyl group, and a lactone ring.Broadly speaking, a compound possessing the ent-gibberellane ring systembut lacking one or more of these structural features can be considered agibberellin precursor, an intermediate, or a derivative.

The compositions and methods of the invention generally employ activeforms of gibberellic acids, gibberellic acid precursors, gibberellicacid intermediates or gibberellic acid derivatives, for example, thosehaving structures related to formulae IV and V as described above.Gibberellins having such structures can have a variety of substituentsincluding hydroxy (—OH), carboxylate (—COOH), ether (—O), methyl (—CH₃),methylene (═CH₂), lactone (—CO—O—) ring, hydroxymethylene (—CH₂—OH),formyl (CHO), and related substituents in a variety of positions. Thegibberellins employed can also have double bonds within the ringstructure at different positions.

In some embodiments, the gibberellic acids can have any of formulae VI,VIa, or VIb.

Gibberellin having formula VI is often referred to as Gibberellin A₃.

Gibberellin having formula VIa is often referred to as Gibberellin A₄.

Gibberellin having formula VIb is often referred to as Gibberellin A₇.

When present in the compositions of the present invention, gibberellicacids or their derivatives can be used in an amount of about 0.001% toabout 50%, or from about 0.01% to about 20%, or from about 0.01% toabout 10%, or from about 0.05% to about 5%, or from about 0.05% to about2% of the composition. According to the invention, in situconcentrations of an active gibberellic acid ranging from about 10⁻⁴ Mto about 10⁻⁶ M are effective for increasing epidermal cellproliferation. Compositions having about 0.5% gibberellic acid are shownherein to promote skin turnover.

Jasmonic Acid Compounds

Jasmonic acid compounds employed in the invention include jasmonic acidand jasmonic acid derivatives available to one of skill in the art. Suchcompounds include jasmonic acid, methyl jasmonate and their isomers. Inthe present invention jasmonic acid and jasmonic acid derivatives usedalso include synthetic and natural stereoisomers of jasmonic acid,dihydrojasmonic acid, hydroxy jasmonic acid and dihydro-hydroxy jasmonicacid. Further examples of jasmonic acid derivatives that may be used inthe invention include compounds having any one of formulae VII, VIII, IXor X.

wherein:

R₄ is alkyl;

R₅ is COOR, or —(CH₂)n-OX, where n is an integer of from 1 to 20;

R is H, or alkyl; and

X is H, or 1 to 6 sugar residues (e.g., hexoses or pentoses).

In general, the alkyl groups employed in these jasmonic acid compoundshave about one to twenty carbon atoms, although in some embodimentslower alkyl groups are used, for example, alkyl groups with about one toeight carbon atoms. Alkyl groups with even lower numbers of carbon atomscan also be used, for example, alkyl groups with one to six, or one tothree carbon atoms.

In some embodiments, jasmonic acid is employed in the compositions ofthe invention. Jasmonic acid is a compound of formula VII wherein R₄ isC₂H₅ and R₅ is COOH.

Another jasmonic acid compound employed in the invention is a compoundof formula VIII

wherein:

R₄ is alkyl;

R₅ is COOR, or —(CH₂)n-OX, where n is an integer of from 1 to 20;

R is H, or alkyl; and

X is H, or 1 to 6 sugar residues (e.g., hexoses or pentoses).

In some embodiments, dihydrojasmonic acid is employed in thecompositions of the invention. Dihydrojasmonic acid is a compound offormula VIII wherein R₄ is C₂H₅ and R₅ is COOH.

Another jasmonic acid compound employed in the invention is a compoundof formula IX

wherein:

R₄ is alkyl;

R₅ is COOR, or —(CH₂)n-OX, where n is an integer of from 1 to 20;

R is H, or alkyl;

X is H, or 1 to 6 sugar residues (e.g., hexoses or pentoses); and

Y is H, alkyl, or 1 to 6 sugar residues (e.g., hexoses or pentoses).

In some embodiments, hydroxy jasmonic acid is employed in thecompositions of the invention. Hydroxy jasmonic acid is a compound offormula IX wherein R₄ is C₂H₅ and R₅ is COOH.

Another jasmonic acid compound employed in the invention is a compoundof formula X.

wherein:

R₄ is alkyl;

R₅ is COOR, or —(CH₂)n-OX, where n is an integer of from 1 to 20;

R is H, or alkyl;

X is H, or 1 to 6 sugar residues (e.g., hexoses or pentoses); and

Y is H, alkyl, or 1 to 6 sugar residues (e.g., hexoses or pentoses).

In some embodiments, dihydro-hydroxyjasmonic acid is employed in thecompositions of the invention. Dihydro-hydroxyjasmonic acid is acompound of formula X wherein R₄ is C₂H₅ and R₅ is COOH.

When present in the compositions of the present invention, jasmonicacids or jasmonic acid derivatives can be used in an amount of fromabout 0.001% to about 50%, or from about 0.01% to about 20%, or fromabout 0.01% to about 10%, or from about 0.05% to about 5%, or from about0.05% to about 2% of the composition. According to the invention, insitu concentrations of jasmonic acid ranging from about 10⁻⁴ M to about10⁻⁶ M are effective for increasing cell proliferation in epidermaltissues. As illustrated herein solutions of about 0.025% jasmonic acidare effective for promoting skin turnover and renewal.

Additional Ingredients

In another embodiment, the compositions and methods of the inventioninclude administering to the female mammal an effective amount ofretinoid or carotenoid and/or one or more nucleotide(s) ornucleoside(s). These compositions and methods can increase theexpression of P2Y₂ receptors or estrogen receptors or vascularendothelial growth factor in vaginal or cervical epithelial cells.

In some embodiments, the compositions of the invention can include oneor more retinoids or carotenoids. The IUPAC-IUB Joint Commission onBiochemical Nomenclature states that “retinoids are a class of compoundsconsisting of four isoprenoid units joined in a head to tail manner.”All retinoids may be formally derived from a monocyclic parent compoundcontaining five carbon-carbon double bonds and a functional group at theterminus of the acyclic portion. The basic retinoid structure can besubdivided into three segments, namely the polar terminal end, theconjugated side chain, and the cyclohexenyl ring. The basic structuresof the most common natural retinoids are called retinol, retinaldehyde,and retinoic acid. However, retinoids of this invention are not limitedto just retinol, retinaldehyde, and retinoic acid. Instead, theretinoids and carotenoids of the invention also include compoundsfalling within Formula XIA or XIB:

wherein:

Z is CH, or N;

R is H or alkyl of 1 to 6 carbons;

m is an integer having the value of 0-5;

n is an integer having the value of 0-2;

r is an integer having the value 0-2;

L is —(C=Z)-NH— or —NH—(C=Z)- where Z is O or S;

Q is a phenyl, naphthyl, pyridyl, thienyl, furyl, pyridazinyl,pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl or pyrazolyl,wherein the phenyl, naphthyl pyridyl, thienyl, furyl, pyridazinyl,pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl or pyrazolylgroup can be substituted with one or two R¹ groups;

W is F, Br, Cl, I, C₁₋₆ alkyl, fluoro-substituted C₁₋₆ alkyl, NO₂, N₃,OH, OCH₂OCH₃, OC₁₋₁₀ alkyl, tetrazol, CN, SO₂C₁₋₆-alkyl,SO₂C₁₋₆-fluoro-substituted alkyl, SO—C₁₋₆ alkyl, CO—C₁₋₆alkyl, COOR₈,phenyl, phenyl itself substituted with a W group other than with phenylor substituted phenyl, with the proviso that when X is CH and r is 0then n is not 0 and at least one W group is not alkyl;

A is (CH₂)_(q) where q is 0-5, lower branched chain alkyl having 3-6carbons, cycloalkyl having 3-6 carbons, alkenyl having 2-6 carbons and 1or 2 double bonds, alkynyl having 2-6 carbons and 1 or 2 triple bonds;and

B is COOH or a pharmaceutically acceptable salt thereof, COOR⁸,CONR⁹R¹⁰, CH₂OH, CH₂OR¹¹, CH₂OCOR¹¹, CHO, CH(OR¹²)₂, CHOR¹³O, COR⁷,CR⁷(OR¹²)₂, CR⁷OR¹³O, where R⁷ is an alkyl, cycloalkyl or alkenyl groupcontaining 1 to 5 carbons, R⁸ is an alkyl group of 1 to 10 carbons ortrimethylsilylalkyl where the alkyl group has 1 to 10 carbons, or acycloalkyl group of 5 to 10 carbons, or R⁸ is phenyl or loweralkylphenyl, R⁹ and R¹⁰ independently are hydrogen, an alkyl group of 1to 10 carbons, or a cycloalkyl group of 5-10 carbons, or phenyl or loweralkylphenyl, R¹¹ is lower alkyl, cycloalkyl, lower alkyl substitutedcycloalkyl, phenyl or lower alkylphenyl, R¹² is lower alkyl, and R¹³ isdivalent alkyl radical of 2-5 carbons.

In some embodiments, the retinoid or carotenoid is a compound defined byFormula XII:

wherein:

R₂₀, R₂₁ and R₂₂ are each independently C₁₋₆ alkyl, fluoro-substitutedC₁₋₆ alkyl, hydroxy-substituted C₁₋₆alkyl, CH₂OH, CH₂OR¹¹, CH₂OCOR¹¹,CHO, CH(OR¹²)₂, CHOR¹³O, COR⁷, CR⁷(OR¹²)₂, or CR⁷OR¹³O.

The term alkyl refers to and covers any and all groups that are known asnormal alkyl, branched-chain alkyl and cycloalkyl. The term alkenylrefers to and covers normal alkenyl, branch chain alkenyl andcycloalkenyl groups having one or more sites of unsaturation. Similarly,the term alkynyl refers to and covers normal alkynyl, and branch chainalkynyl groups having one or more triple bonds.

Lower alkyl means alkyl groups having 1 to 6 carbons, and 3 to 6 carbonsfor lower branch chained and cycloalkyl groups. Lower alkenyl is definedsimilarly having 2 to 6 carbons for normal lower alkenyl groups, and 3to 6 carbons for branch chained and cyclo-lower alkenyl groups. Loweralkynyl is also defined similarly, having 2 to 6 carbons for normallower alkynyl groups, and 4 to 6 carbons for branch chained loweralkynyl groups.

Compounds of Formula XIA, XIB and XII can be made as described in U.S.Pat. No. 6,437,129 and U.S. Pat. No. 6,437,003, which are incorporatedherein in their entirety.

A pharmaceutically acceptable salt may be prepared for any compounds inthis invention having a functionality capable of forming such a salt,for example, an acid or amine functionality. A pharmaceuticallyacceptable salt is any salt that retains the activity of the parentcompound and does not impart any deleterious or untoward effect on thesubject to which it is administered and in the context in which it isadministered. Pharmaceutically acceptable salts may be derived fromorganic or inorganic bases. The salt may be a mono or polyvalent ion. Ofparticular interest are the inorganic ions, sodium, potassium, calcium,and magnesium. Organic salts may by be made with amines, particularlyammonium salts such as mono-, di- and trialkyl amines or ethanolamines.Salts may also be formed with caffeine, tromethamine and similarmolecules. Where there is a nitrogen sufficiently basic as to be capableof forming acid addition salts, such may be formed with any inorganic ororganic acids or alkylating agent such as methyl iodide. Preferred saltsare those formed with inorganic acids such as hydrochloric acid,sulfuric acid or phosphoric acid. Any of a number of simple organicacids such as mono-, di- or tri-acid may also be used.

Some of the compounds of the present invention may have trans and cisisomers. In addition, the compounds of the present invention may containone or more chiral centers and therefore may exist in enantiomeric anddiastereomeric forms. The scope of the present invention is intended tocover all such isomers, as well as mixtures of cis and trans isomers,mixtures of diastereomers and racemic mixtures of enantiomers (opticalisomers) as well.

The compositions of the invention can include one or more nucleosides ornucleotides. Such nucleotide or nucleosides can include, for example,dATP, dGTP, dCTP, dTTP, dUTP, ATP, GTP, CTP, TTP, UTP, and anyderivative of such nucleotide(s) or nucleoside(s) that is available toone of skill in the art.

In some embodiments, the nucleotide or nucleoside is a compound definedby Formula XIII:

wherein:

X₁, X₂ and X₃ are each independently either O⁻ or S⁻. In someembodiments, X₂ and X₃ are each O⁻;

R₃₁ is O, imido, methylene, or dihalomethylene (e.g., dichloromethylene,difluoromethylene). In some embodiments, R₃₁ is oxygen ordifluoromethylene.

R₃₂ is H or Br. In some embodiments, R₃₂ is H. Examples of compounds ofFormula XIII are uridine 5′-triphosphate (UTP) and uridine5′-O-(3-thiotriphosphate) (UTPγS).

In other embodiments, the nucleotide or nucleoside is a compound definedby Formula XIV:

wherein V is uracil or adenine.

In further embodiments of the invention, the nucleotide or nucleoside isa compound defined by Formula XV:

wherein:

X₁, X₂, X₃ and R₃, are as defined above;

R₃₃ and R₃₄ are H when R₃₅ is nothing and there is a double bond betweenN-1 and C-6 (adenine), or R₃₃ and R₃₄ are H when R₃₅ is O and there is adouble bond between N-1 and C-6 (adenine 1-oxide), or R₃₃, R₃₄ and R₃₅taken together are —CH═CH—, forming a ring from N-6 to N-1 with a doublebond between N-6 and C-6 (1,N⁶-ethenoadenine).

In still further embodiments of the invention, the nucleotide ornucleoside is a compound defined by Formula XVI:

wherein:

X₁, X₂, X₃ and R₃₁ are as defined above;

R₃₆ and R₃₇ are H when R₃₈ is nothing and there is a double bond betweenN-3 and C-4 (cytosine), or, R₃₆, R₃₇ and R₃₈ taken together are —CH═CH—,and form a ring from N-3 to the nitrogen attached to R₃₆ and R₃₇(3,N⁴-ethenocytosine).

Hence, compositions of the invention can contain one or more compoundsof Formula XIII, XIV, XV or XVI in an amount effective to stimulatemucous secretions in the vagina or reproductive passages of a female.

Compounds illustrative of the compounds of Formula XIII above include:(a) uridine 5′-triphosphate (UTP); (b) uridine 5′-O-(3-thiotriphosphate)(UTPγS); and (c) 5-bromo-uridine 5′-triphosphate (5-BrUTP). Thesecompounds are known or may be made in accordance with known procedures,or variations thereof which will be apparent to those skilled in theart. See generally N. Cusack and S. Hourani, Annals N.Y. Acad. Sci. 603,172-81 (entitled “Biological Actions of Extracellular ATP”). Forexample, UTP may be made in the manner described in Kenner, et al., J.Chem. Soc. 1954, 2288; or Hall and Khorana, J. Am. Chem. Soc. 76, 5056(1954). See Merck Index, Monograph No. 9795 (11th Ed. 1989). UTPγS maybe made in the manner described in R. S. Goody and F. Eckstein, J. Am.Chem. Soc. 93, 6252 (1971).

Compounds illustrative of the compounds of Formula XIV includeP¹,P⁴-di(adenosine-5′) tetraphosphate or P¹,P⁴-di(uridine-5′)tetraphosphate. These compounds can be made in accordance with knownprocedures, or variations thereof which will be described by: P.Zamecnik, et al., Proc. Natl. Acad. Sci. USA 89, 838-42 (1981); and K.Ng and L. E. Orgel, Nucleic Acids Res. 15 (8), 3572-80 (1987). P¹,P⁴-di(uridine-5′) tetraphosphate can be prepared by methods similar tothat described in C. Vallejo, et al., Biochem. Biophys. Acta 438, 304-09(1976).

Compounds illustrative of the compounds of Formula XV above include (a)adenosine 5′-triphosphate (ATP) and (b) 1,N⁶-ethenoadenosine5′-triphosphate. Compounds illustrative of the compounds of Formula XIVabove include (a) cytidine 5′-triphosphate and (b) 3,N⁴-ethenocytidine5′-triphosphate. These compounds can be made in accordance with knownprocedures, or variations thereof which will be apparent to thoseskilled in the art. For example, phosphorylation of nucleosides bystandard methods such as D. Hoard and D. Ott, J. Am. Chem. Soc. 87,1785-1788 (1965); M. Yoshikawa, et al., Tetrahedron Lett. 5065-68 (1967)and idem., Bull. Chem. Soc. (Jpn) 42, 3505-08 (1969); J. Moffatt and H.Khorana, J. Am. Chem. Soc. 83, 649-59 (1961); and B. Fischer, et al., J.Med. Chem. 36, 3937-46 (1993) and references therein. Etheno derivativesof cytidine and adenosine are prepared by known methods such as: N.Kotchetkov, et al., Tetrahedron Lett. 1993 (1971); J. Barrio, et al.,Biochem. Biophys. Res. Commun. 46, 597 (1972); J. Secrist, et al.,Biochemistry 11, 3499 (1972); J. Bierndt, et al., Nucleic Acids Res. 5,789 (1978); K. Koyasuga-Mikado, et al., Chem. Pharm. Bull. (Tokyo) 28,932 (1980). Derivatives with alpha, beta and gamma thiophosphorus groupscan be derived by the following or by adapting methods of: J. Ludwig andF. Eckstein, J. Org. Chem. 54, 631-35 (1989); F. Eckstein and R. Goody,Biochemistry 15, 1685 (1976); R. Goody and F. Eckstein, J. Am. Chem.Soc. 93, 6252 (1971).

Compounds of Formulas XIII, XV or XVI where R₁ is CCl₂ and CF₂ can beprepared by methods similar to that described in G. Blackburn, et al.,J. Chem. Soc. Perkin Trans. 1, 1119-25 (1984). Compounds of Formula I,II, III where R₁ is CH₂ can be prepared by methods similar to thatdescribed in T. Myers, et al., J. Am. Chem. Soc. 85, 3292-95 (1963).

In addition, UTP, ATP, CTP, P¹P⁴-di(adenosine-5′) tetraphosphate,3,N⁴-ethenocytidine triphosphate, 1,N⁶-ethenoadenine 5′-triphosphate,adenosine 1-oxide 5′-triphosphate, ATPγS, ATPβS, ATPαS, AMPPCH₂—P,AMPPNHP, N⁴-ethenocytidine and 1,N⁶-ethenoadenosine are commerciallyavailable, for example, from Sigma Chemical Company, PO Box 14508, St.Louis, Mo. 63178.

Methods of Use

The present invention is directed to a variety of methods of treating orpreventing vaginal and/or reproductive problems in a female mammal.These methods involve administering to the female mammal an effectiveamount of vitamin E and/or a plant hormone such as gibberellic acid orjasmonic acid. In general, administration is topical or intravaginal.

Treatment of, or treating, vaginal and/or reproductive problems in afemale mammal is intended to include modulation of mucus levels toenhance or diminish fertility in a female mammal, or to alleviate ordiminish vaginal dryness in a female mammal. The treatment therefore caninclude alleviation or diminishment of more than one vaginal and/orreproductive problem in a female mammal.

In one embodiment, the method increases growth of vaginal or cervicalepidermal cells. In another embodiment, the method involves increasingthe expression of mucin genes, for example, mucin-1, mucin-2, mucin-3A,mucin-3B, mucin-4, mucin-5B, mucin-5AC, mucin-6, mucin-7, mucin-11,mucin-13, mucin-15, mucin-17, mucin-19, mucin-20 and similar mucingenes. Such methods can prevent or treat vaginal dryness in a mammal, ormaintain or enhance the normal protective function of vaginal mucus in amammal.

The term “mammal,” as used herein, refers to an animal, in general, awarm-blooded animal. Mammals include cattle, buffalo, sheep, goats,pigs, horses, dogs, cats, rats, rabbits, mice, and humans. Also includedare other livestock, domesticated animals and captive animals.

Treatment involves administering an effective amount of vitamin E and/ora plant hormone. The vitamin E, gibberellic acid and/or jasmonic acidmay be administered as a composition that contains other ingredients,for example, one or more nucleotide(s) or nucleoside(s), other vitamins(e.g., a retinoid or carotenoid such as vitamin A), aloe vera and thelike. In general, compositions containing vitamin E, gibberellic acid orjasmonic acid with or without other compound(s) are administeredintravaginally.

Compositions

The compositions of the invention are administered to improve the healthof the female reproductive system, to stimulate secretion of lubricatingfluids (mucus), promote cell growth, treat regression of vaginal mucosaand rejuvenate the female reproductive system.

To achieve the desired effect(s), the composition may be administered assingle or divided dosages, for example, of at least about 0.001 μg/kg toabout 100 to 200 mg/kg, of about 0.01 μg/kg to about 30 to 50 mg/kg,about 0.1 μg/kg to about 10 to 20 mg/kg or about 1.0 μg/kg to about 1.0to about 10 mg/kg of body weight of one or more retinoid or carotenoidor nucleotide or nucleoside, although other dosages may providebeneficial results. The amount administered will vary depending onvarious factors including, but not limited to, the disease, the weight,the physical condition, the health, the age of the mammal, and whetherprevention of reproduction or treatment of vaginal dryness is to beachieved. Such factors can be readily determined by the clinicianemploying animal models or other test systems that are available in theart.

Administration of the therapeutic agents in accordance with the presentinvention may be in a single dose, in multiple doses, in a continuous orintermittent manner, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of the compositions of the inventionmay be essentially continuous over a pre-selected period of time or maybe in a series of spaced doses. Local administration is generallycontemplated.

The compositions are prepared by combining the active ingredients in theappropriate concentrations. Other active or inactive agents selected byone of skill in the art can optionally be added. The absolute weight ofa given active agent included in a unit dose can vary widely. Forexample, about 0.001 μg to about 50 mg, or about 0.01 μg to about 10 mg,or about 0.1 μg to about 1 mg, of at least one vitamin E, gibberellicacid or jasmonic acid, or of a plurality of vitamin E compounds orgibberellic acid compounds or jasmonic acid compounds can beadministered. Alternatively, the unit dosage can vary from about 0.001μg to about 1000 μg, from about 0.01 μg to about 750 μg, from about 0.1μg to about 1 mg, from about 1.0 μg to about 750 μg, from about 2.5 μgto about 600 μg, from about 5.0 μg to about 500 μg, or from about 7.5 μgto about 400 μg.

Daily doses of the compositions of the invention can vary as well. Suchdaily doses can range, for example, from about 0.001 mg/day to about 50mg/day, from about 0.01 mg/day to about 25 mg/day, from about 0.1 mg/dayto about 12 mg/day, from about 0.1 mg/day to about 8 mg/day, from about0.1 mg/day to about 4 mg/day, and from about 0.1 mg/day to about 2mg/day of one or more vitamin E, gibberellic acid or jasmonic acidcompounds.

The concentration of vitamin E, gibberellic acid or jasmonic acid withina composition can also vary. For example, the concentration can varyfrom about 0.1 μM to about 1000 μM, or from about 0.5 μM to about 500μM, or from about 1 μM to about 300 μM, or from about 5 μM to about 200μM, or from about 10 μM to about 100 μM.

Thus, one or more suitable unit dosage forms comprising the therapeuticcompositions of the invention can be administered by a variety of routesincluding oral, parenteral (including subcutaneous, intravenous,intramuscular and intraperitoneal), rectal, dermal, transdermal,intrathoracic, intrapulmonary intravaginal and intranasal (respiratory)routes. The therapeutic compositions may also be formulated forsustained release (for example, using microencapsulation, see WO94/07529, and U.S. Pat. No. 4,962,091). The formulations may, whereappropriate, be conveniently presented in discrete unit dosage forms andmay be prepared by any of the methods well known to the pharmaceuticalarts. Such methods may include the step of mixing the therapeutic agentwith liquid carriers, solid matrices, semi-solid carriers, finelydivided solid carriers or combinations thereof, and then, if necessary,introducing or shaping the product into the desired delivery system.

When the therapeutic compositions of the invention are prepared forintravaginal administration, they are generally combined with apharmaceutically acceptable carrier, diluent or excipient to form apharmaceutical formulation, or unit dosage form. For intravaginaladministration, the compositions may be present as a solution, asuspension, an emulsion, a powder, a granular formulation, or in anatural or synthetic polymer or resin. The active compositions may alsobe presented as a bolus or paste. Intra vaginally administeredtherapeutic compositions of the invention can also be formulated forsustained release, e.g., the compositions can be coated,micro-encapsulated, or otherwise placed within a sustained deliverydevice. The total active ingredients in such formulations comprise from0.1 to 99.9% by weight of the formulation.

By “pharmaceutically acceptable” it is meant a carrier, diluent,excipient, and/or salt that is compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof.

Pharmaceutical formulations containing the therapeutic compositions ofthe invention can be prepared by procedures known in the art usingwell-known and readily available ingredients. For example, thecomposition can be formulated with common excipients, diluents, orcarriers, and formed into tablets, capsules, solutions, suspensions,powders, aerosols and the like. Examples of excipients, diluents, andcarriers that are suitable for such formulations include buffers, aswell as fillers and extenders such as starch, cellulose, sugars,mannitol, and silicic derivatives. Binding agents can also be includedsuch as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose and other cellulose derivatives, alginates, gelatin, andpolyvinylpyrrolidone. Moisturizing agents can be included such asglycerol, disintegrating agents such as calcium carbonate and sodiumbicarbonate. Agents for retarding dissolution can also be included suchas paraffin. Resorption accelerators such as quaternary ammoniumcompounds can also be included. Surface active agents such as cetylalcohol and glycerol monostearate can be included. Adsorptive carrierssuch as kaolin and bentonite can be added. Lubricants such as talc,calcium and magnesium stearate, and solid polyethyl glycols can also beincluded. Preservatives may also be added. The compositions of theinvention can also contain thickening agents such as cellulose and/orcellulose derivatives. They may also contain gums such as xanthan, guaror carbo gum or gum arabic, or alternatively polyethylene glycols,bentones and montmorillonites, and the like. The compositions of theinvention can also contain other vitamins such as vitamin A, vitamin B,vitamin C or vitamin D. In one embodiment, the compositions of theinvention can also contain aloe vera.

The therapeutic compositions of the invention can also be formulated asemulsions, suspensions, aqueous or anhydrous solutions or dispersions,or alternatively the form of an emulsion or suspension or salve forconvenient intravaginal administration. The active compositions andother ingredients may form suspensions, solutions, or emulsions in oilyor aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive compositions and other ingredients may be in powder form,obtained by aseptic isolation of sterile solid or by lyophilization fromsolution, for constitution with a suitable vehicle, e.g., sterile,pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable carriers,vehicles and adjuvants that are well known in the art. It is possible,for example, to prepare solutions using one or more organic solvent(s)that is/are acceptable from the physiological standpoint, chosen, inaddition to water, from solvents such as acetone, ethanol, isopropylalcohol, glycol ethers such as the products sold under the name“Dowanol,” polyglycols and polyethylene glycols, C₁-C₄ alkyl esters ofshort-chain acids, ethyl or isopropyl lactate, fatty acid triglyceridessuch as the products marketed under the name “Miglyol,” isopropylmyristate, animal, mineral and vegetable oils and polysiloxanes.

It is possible to add, if necessary, an adjuvant chosen fromantioxidants, surfactants, other preservatives, film-forming,keratolytic or comedolytic agents, perfumes, flavorings and colorings.Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole,butylated hydroxytoluene and α-tocopherol and its derivatives can beadded.

Additionally, the compositions are well suited to formulation assustained release dosage forms and the like. The formulations can be soconstituted that they release the active composition within the femalereproductive system over a period of time. Coatings, envelopes, andprotective matrices may be made, for example, from polymeric substances,such as polylactide-glycolates, liposomes, microemulsions,microparticles, nanoparticles, or waxes.

For intravaginal administration, the therapeutic agents may beformulated as is known in the art for direct application to the vaginalarea. Forms chiefly conditioned for vaginal application take the form,for example, of creams, milks, gels, dispersion or microemulsions,lotions thickened to a greater or lesser extent, impregnated pads,ointments, aerosol formulations (e.g., sprays or foams), creams,lotions, pastes, jellies, sprays, and aerosols. Alternatively, thecomposition can be formulated to be part of an adhesive polymer, such aspolyacrylate or acrylate/vinyl acetate copolymer.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. Liquid sprays are conveniently delivered frompressurized packs, for example, via a specially shaped closure. Theactive compositions can also be delivered via iontophoresis, e.g., asdisclosed in U.S. Pat. No. 4,140,122; 4,383,529; or 4,051,842. Thepercent by weight of a therapeutic agent of the invention present in avaginal formulation will depend on various factors, but generally willbe from 0.01% to 95% of the total weight of the formulation, andtypically 0.1-85% by weight.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that areavailable in the art. Examples of such substances include normal salinesolutions such as physiologically buffered saline solutions and water.Specific non-limiting examples of the carriers and/or diluents that areuseful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable buffered saline solutionssuch as phosphate buffered saline solutions with a pH of about 4.5 toabout 5.5.

Furthermore, the active ingredients may also be used in combination withother therapeutic agents, for example, anti-microbial agents, painrelievers, anti-inflammatory agents, vitamins (e.g., vitamin A, B, C orD), aloe vera and the like, whether for the conditions described or someother condition.

The present invention further pertains to a packaged pharmaceuticalcomposition for controlling reproduction and/or vaginal dryness such asa kit or other container. The kit or container holds a therapeuticallyeffective amount of a pharmaceutical composition for controllingreproduction and/or vaginal dryness and instructions for using thepharmaceutical composition for control of reproduction and/or vaginaldryness. The pharmaceutical composition includes a composition of thepresent invention, in a therapeutically effective amount such thatvaginal dryness is controlled.

In addition, the invention provides a vaginal insert that can releasethe vitamin E, gibberellic acid or jasmonic acid in a controlledfashion. Such a vaginal insert can be biodegradable ornon-biodegradable. The vaginal insert provides sustained release of theactive ingredients at an appropriate rate for achieving the desiredlevel of mucus secretion, gene expression and cellular proliferation.

In some embodiments, the active ingredients can be formulated witholeaginous bases or ointments to form the vaginal insert. This class offormulations comprises the active ingredients and hydrocarbon-basedsemisolids containing dissolved and/or suspendedbacteriostats/preservatives and a buffer system. The petrolatumcomponent in these bases can be any paraffin ranging in viscosity frommineral oil employing incorporated isobutylene, colloidal silica, orstearate salts to paraffin waxes. White and yellow petrolatum areexamples of such systems. Bases of this class can be made byincorporating high-melting waxes into a fluid mineral oil via fusion orby incorporation of polyethylene into mineral oil at elevatedtemperature. Polysiloxanes (also known as silicones) are suitable foruse in these bases and typically have a viscosity in the range of about0.5 to 10.sup.6 centistokes. The organic entities attached to thepolysiloxane are preferably lower molecular weight hydrocarbon moietieshaving from 1 to 8 carbons each, such as lower alkyl, lower alkenyl,phenyl and alkyl substituted phenyl, and phenyl(lower)alkyl, such asbenzyl. In such a moiety, each lower alkyl or alkenyl group preferablyhas 1 to 3 carbons inclusive, such as in a dimethylsiloxane polymer.

Absorption bases can used with such an oleaginous system. In addition tothe active ingredients, additional ingredients with the capacity toemulsify a significant quantity of water are employed. Water-in-oil(w/o) emulsions can be formed wherein the external phase is oleaginousin character. Preservatives/bacteriostats, such as the parabens, buffersystems, etc. can be incorporated into these bases as emulsified aqueoussolutions together with the active ingredient. Diverse additives areconveniently used as the emulsifier, and these include, but are notlimited to, cholesterol, lanolin (which contains cholesterol andcholesterol esters and other emulsifiers), lanolin derivatives, beeswax,fatty alcohols, wool wax alcohols, low HLB (hydrophobe/lipophobebalance) emulsifiers, and assorted ionic and nonionic surfactants,singularly or in combination.

Water-In-Oil (W/O) emulsion bases can be employed in the vaginal insertsof the invention. These formulations can be an expansion of the generalclass of absorption bases that includes liquids or creams. They can beprepared by taking a mixture of the active ingredients with oil phaseingredients, bacteriostats/preservatives and buffer salts which aredissolved or suspended therein and to which water has been added to forma water-in-oil emulsion.

Oil-In-Water (O/W) emulsion bases can also be utilized in the vaginalinserts of the invention. These systems are semisolid emulsions,microemulsions, or foam emulsion systems containing metronidazole.Usually such a system has a “creamy white” appearance. Typically, theinternal oil phase is in the range in percentage composition of about10% to about 40% oil by weight and the external phase may contain 80% ormore water. The oleaginous phase may contain, but is not limited to,long-chain alcohols (cetyl, stearyl), long-chain esters (myristates,palmitates, stearates), long-chain acids (palmitic, stearic), vegetableand animal oils and assorted waxes. These can be made with anionic,cationic, nonionic or amphoteric surfactants, or with combinationsespecially of the nonionic surfactants. The examples below are exemplaryof these systems, but those skilled in the art will appreciate thatsubstitutions and additions or omissions of the specified componentscould be made by one who is skilled in the art.

Vaginal inserts and suppositories containing the active ingredients canbe, for example, oleaginous in nature that melt at body temperature, orpolyethylene glycol-based compositions that dissolve in the vaginalfluids. Additional bases for suppositories are glycerin and glycerinatedgelatin.

The active ingredients can also be formulated into vaginal inserts usingbuffered gels made with gelling agents. Some examples of these gellingagents are: cellulosics, cationic polymers, polyoxyalkylenes, andcarboxyvinyl polymers. Cellulosics useful in the formulations of theinvention include, for example, methyl cellulose, carboxymethylcellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose. CationicPolymers useful in the formulations of the invention include“Polyquaternium-10”, a polymeric quaternary ammonium salt ofhydroxyethyl cellulose reacted with a trimethyl ammonium-substitutedepoxide, and the like. Polyoxyalkylenes useful in the invention includeolyoxyethylene-polyoxypropylene esters of lanolin and derivativesthereof. Carboxyvinyl polymers useful for the formulations of theinvention include cross-linked acrylic acid polymers, e.g., thosecommercially available from B.F. Goodrich Co., Akron, Ohio, under thedesignation CARBOPOL™.

The vaginal insert can comprise a mixture or coating of polymers thatprovide release of the active agents at a constant rate over a prolongedperiod of time. In some embodiments, the vaginal insert compriseswater-soluble pore forming agents, such as polyethylene glycol (PEG)that can be mixed with water insoluble polymers to increase thedurability of the insert and to prolong the release of the activeingredients. Such a water-soluble pore forming agent can be polyethyleneglycol, polypropylene glycol, a mixture or polymer of sugars (lactose,sucrose, dextrose, etc.), salts, poloxamers, polyvinyl alcohol and otherwater soluble food grade and other excipients.

When PEG is used as a pore forming agent, the molecular weight of PEG isin the range from about 200 to about 20,000, alternatively, from about400 to about 8,000. For example, PEG having a molecular weight of about540 to about 8,000 is used. In another embodiment, the PEG has amolecular weight of about or above 1,000 to about 8,000. The molecularweight of PEG used for the coating with the formulation of the inventionwill depend on the ability of PEG to form a coating film that isnon-sticky, having enough strength and creating adequate pore size forcontrolling the release of active ingredients over the desired timeperiod both in vitro and in vivo.

The pore-forming agent is used in the formulation of the invention inthe amount effective to regulate the release of a biologically activecompound at a desired rate. Preferably, the effective amount of thepore-forming agent provides long term delivery of the active agent thusincreasing the useful life of a sustained-release drug implant. Theeffective amount of the pore forming agent will depend on the desiredrate and duration of the release and the ability to form a continuousmicroporous film during the coating process. To enable release durationover longer periods of time PEG with higher molecular weights is used.For example, PEG 8000 can provide release over a period of time that islonger than 100 days, when used in a concentration from 10 to 50%,preferably from 20 to 45% and most preferably from 30 to 45%. Theconcentration of PEG is expressed herein in % weight per dry basis andrepresents the concentration of PEG in the coating film after drying.Similarly, the thickness of the coating film is from 5 to 50 μm,preferably 30 from 10 to 30 μm and most preferably from 15 to 25 μm.

A good correlation exists between the dissolution rate of active agentsand the amount of pore forming agent incorporated in the coating filmbased on in vitro and in vivo studies shown in the Examples. Dependingon the desired length of release, the PEG concentration ranges can beadjusted as needed. For example, in vivo duration of a coated insert maybe predicted simply from the in vitro dissolution rate of the activeagent at the 120-hour time point.

The vaginal insert of the invention may also comprise a water insolublepolymer. Examples of such polymers are ethylcellulose, acrylic resins,co-polymer of methacrylic acid and acylic acid ethyl ester, polylacticacid, PLGA, polyurethane, polyethylene vinyl acetate copolymer,polystyrene-butadiene copolymer and silicone rubber, or mixturesthereof. For example, polymers sold under trade names Aquacoat ECD 30and Eudragit RS 30 and NE 30D (registered trademarks of Rhom Tech, Inc.)can be used.

A polymer suitable for use in this invention is a polymer that iscapable of forming a continuous coating film during the process ofspraying and drying with a pore-forming agent. The rate controlling filmprepared with such a polymer is very stable during implantation. Thefilm should have enough strength to withstand tear and inner osmoticpressure, and have the stability not to swell or hydrate during theimplantation life.

In one embodiment, the coating formulation of the invention is used tocoat pellets comprising the active ingredients that are compressed toform a solid, biodegradable insert and then administered for stimulatingmucus secretion, gene expression and/or cellular proliferation.

The invention is further illustrated by the following non-limitingExamples.

Example 1 Vitamin E Increases Vaginal Cell Growth and Mucus Secretion

This Example shows that vitamin E can enhance human cervical epithelialcell growth and increase the expression of mucin-4 in an atrophicvaginitis model.

Materials and Methods

Cell Culture. Human cervical epithelial cells (ME-180, ATCC) werepropagated in culture at 37° C. using 5% CO₂ in McCoy's 5A media(Invitrogen) with 10% heat-inactivated fetal bovine serum (Invitrogen).Vitamin E was purchased from ICN Biomedicals. Vitamin E stock solutionswere made up in absolute ethanol.

RNA Extraction from ME-180 cells. ME-180 cells were plated at 2×10⁵cells per T75 flask (15 mL total volume) and were allowed to propagatefor four days. On day four, the media was removed. Fifteen mL of McCoy'sSA media with or without 10% FBS containing 0 or 1 μM vitamin E wereadded to ME-180 cells in duplicate. ME-180 cells were incubated withthese solutions for 24 hours. The media was removed, and 1500 μL oflysis buffer was added to the flasks. The cells were scraped off of theflask with a cell scraper. The RNA was isolated using the AmbionRNAqueous kit according to the manufacturer's instructions. PrecipitatedRNA was resuspended in DEPC-treated water with 1 μL of RNase inhibitor.

Reverse Transcription of RNA. Approximately 7 μL of RNA was incubatedwith 3 μL of primer at 80° C. for 10 minutes then immediately placed onice. A master mix containing 4 μL DEPC water, 4 μL 5× reaction buffer, 1μL dNTPs, 1 μL of RNase inhibitor, and 1 μL of reverse transcriptase wasmade for the reactions. Ten μL of master mix was added to the RNA/primersolution and placed at 42° C. for 2 hours. The resulting cDNA wasincubated with 3.5 μL of NaOH solution at 70° C. for 10 minutes tohydrolyze the remaining RNA. Five μL of 0.5M Tris/EDTA was added toneutralize the solution. To precipitate the cDNA, 125 μL of 3M NH₄OAc, 5μL of linear polyacrylamide, and 700 μL of absolute ethanol were added.The solutions were vortexed and placed at −20° C. overnight. Sampleswere centrifuged at 14000 rpm at 4° C. for 30 minutes. The ethanol wasremoved, and the samples were allowed to dry for 10 minutes. The cDNAwas resuspended in 10-12 μL of PCR grade water.

Polymerase Chain Reaction (PCR). A master mix using the Advantage 2 PCRkit (Ambion) was made for the PCR reactions containing 37-38 μL of PCRgrade water, 5 μL of 10×PCR buffer, 1 μL of dNTPs, 1 μL of β-actinprimers (10 μM, optional control), and 2 μL of test primers (10 μM,P2Y₂, or Mucin-4 (MUC-4)). Two microliters of cDNA and 1 mL of DNApolymerase were added. Cycling conditions were as follows: 5 min at 95°C.; 25-30 cycles (30 sec at 95° C., 1 min at 65° C., 3 min at 68° C.); 5min at 68° C.

The PCR primers employed were as follows:

P2Y₂-specific primers (yielding a PCR product of 650 bp): (SEQ ID NO: 1)coding strand: 5′-TGTCTTCGCCCTCTGCTTCC-3′ (SEQ ID NO: 2) noncodingstrand: 5′-GTCAGGCCAGGGGTGTCATT-3′ β-actin-specific primers (PCR product300 bp): (SEQ ID NO: 3) coding strand: 5′-AGTCGGTTGGAGCGAGCATC-3′ (SEQID NO: 4) noncoding strand: 5′-GGGCACGAAGGCTCATCATT-3′ Mucin-4 (MUC-4)specific primers (PCR product 800 bp): (SEQ ID NO: 5) codingstrand:5′-AGCCCAGGACTGTGGTCTGC-3′ (SEQ ID NO:6) noncoding strand:5′-GCTCACGTTCAGGGCTGTCA-3′

Results

ME-180 cells were incubated with vitamin E at various concentrations forthree days. At 10 μM vitamin E, ME-180 cell growth was inhibited (datanot shown). However, this may be due to the presence of a largerconcentration of ethanol (used as a solvent for vitamin E) in the media.The negative control with the same amount of ethanol (no vitamin E) alsoshowed no ME-180 cell growth. At 1 μM vitamin E, ME-180 cell growth wasenhanced more than the corresponding negative control. At 100 nM vitaminE, cell growth was not effected in comparison to the negative control.Therefore, a concentration of 1 μM vitamin E was used to measure changesin RNA expression over a 24 hour period.

ME-180 cells were treated with 0 or 1 μM vitamin E in the presence andabsence of fetal bovine serum. Previous results by the inventors hadshown that few changes in RNA expression occur in the presence of serum.However, when the serum was removed, ME-180 cell growth slowed down, aneffect that is observed in vaginal atrophy. Therefore, ME-180 cellsmaintained without serum was used as a model of atrophic vaginitis.

After treatment with vitamin E for 24 hours, RNA was isolated from theME-180 cells. Using reverse transcription, the RNA was converted to cDNAthat was then used as template in PCR reactions. In the presence ofserum, no significant changes in expression of P2Y2 or mucin-4 wereobserved, as had been expected (data not shown).

After 24 hour treatment with vitamin E in the absence of serum, mucin-4expression in ME-180 cells was significantly increased (FIG. 1).Treatment with vitamin E for 24 hours in the absence of serum led to noobservable difference in P2Y2 expression, DTR expression, or VEGFexpression at the 24 time point (data not shown). However, previousexperiments with vitamin A had indicated that after approximately 24hours in the absence of serum, ME-180 cells begin to express P2Y2.Therefore, it is possible that changes occurred during or after the 24hour incubation period that were not detected in this experiment.

These data indicate that vitamin E can enhance human cervical epithelialcell growth as well as increase the expression of mucin-4 in an atrophicvaginitis model. Therefore, vitamin E may be useful in the treatment ofatrophic vaginitis.

Example 2 Vitamin A Effects Cervical Cell Growth and Gene Expression

The interaction of ME-180 cervical cells with several concentrations ofvitamin A derivatives were studied to determine if the vitamin Aderivatives would affect ME-180 cellular growth and gene expression.

Cell Proliferation of ME-180 Cells. ME-180 cells were incubated withvitamin A derivatives for a period of up to 3 days. ME-180 cells werecounted on each day. The results of these experiments are shown in FIGS.2 and 3.

When very low concentrations of vitamin A were used little effect oncell growth was observed. For example, concentrations of 0.0 nM(negative control), 1 nM, 10 nM, and 100 nM trans-retinoic acid,9-cis-retinoic acid or 13-cis-retinoic acid had little effect on cellgrowth. However, cells exposed to trans-retinoic acid appeared to begrowing slightly faster than cells exposed to other agents or to noagents (unexposed, negative control cells).

Therefore, another experiment using retinoic acid concentrations rangingfrom 100 nM to 10 μM was performed. As illustrated in FIG. 2, more cellswere present after treatment with 1 μM or 10 μM trans-retinoic acid thanafter treatment with no retinoic acid (negative control). These dataindicate that trans-retinoic acid enhances ME-180 cell growth inculture. In contrast, cells treated with 13-cis-retinoic acid did notgrow as well as the negative control, indicating that 13-cis-retinoicacid is toxic to ME-180 cells at micromolar concentrations.

Expression Assay Using Polymerase Chain Reaction and ME-180 cDNA. RNAwas isolated from ME-180 cells that had been treated with 1 μM or 10 nMtrans-retinoic acid (vitamin A) for 24 hours. After precipitation, theRNA was used as a template for DNA synthesis by reverse transcription asdescribed above. PCR techniques were then used to amplify the newlysynthesized DNA to determine the approximate level of transcription ofthe P2Y₂ receptor gene (an approximate 600 bp PCR product).“House-keeping genes” or genes that remain at a constant expressionlevel were also amplified by PCR for comparison. The housekeeping genestested with P2Y₂ was β-actin (˜300 bp product).

As shown in FIG. 3, addition of control β-actin primers leads tosynthesis of an approximate 300 bp cDNA product that is present inapproximately equal amounts in all samples. This result indicates thatapproximately the same amounts of template RNA were present in allsamples. However, as is also illustrated in FIG. 3, the P2Y₂ product canbarely be seen in lanes 2, 3, and 7 where the PCR template was cervicalcDNA control, ME-180 cells without vitamin A, and ME-180 cells withoutvitamin A or serum, respectively. Thus, in the absence of vitamin A,little P2Y₂ mRNA is present in ME-180 cells.

However, upon addition of 100 nM vitamin A to ME-180 cells, an increasein P2Y₂ expression is observed both in the presence of serum (FIG. 3,lanes 4-6) and in the absence of serum (FIG. 3, lanes 8-11) in themedia. These data indicate that low concentrations of vitamin A may beuseful in the treatment of atrophic vaginitis.

Cell samples exposed to vitamin A in the absence of serum were alsotested for expression of other genes that may also affect atrophicvaginitis. Using primers designed to amplify the estrogen receptor alpha(ER-α) gene, PCR was performed using the RNA samples from cells starvedof serum. The ER-α product (650 bp) was visible in the control (FIG. 4,lane 2), where the template was cDNA from ME-180 cells cultured in thepresence of serum. In samples that did not contain serum or vitamin A,the cDNA fragment signifying ER-α gene expression was not detected(lanes 3 and 7, FIG. 4). However, in the samples that were exposed to100 nM vitamin A but no serum for 4, 8, and 16 hours, the E

-α gene was expressed (Lanes 4-6, respectively). These data indicatethat vitamin A can induce the expression of E

-α in cervical epithelial cells cultured in the absence of serum.Increasing the amount of estrogen receptors on epithelial cells in thegenital tract may increase the probability that a receptor will beactivated by estrogen.

Moreover, treatment of ME-180 cells with 100 nM vitamin A also increasedthe expression of vascular endothelial growth factor (VEGF), animportant factor involved in blood vessel formation. As illustrated inFIG. 5, an approximate 700 bp product characteristic of VEGF is detectedin ME-180 cells treated with 100 nM vitamin A.

These experiments indicate that vitamin A (trans-retinoic acid) isnontoxic and may be useful for enhancing natural vaginal moisture forwomen suffering from atrophic vaginitis. Vitamin A increases cervicalcell growth at micromolar concentrations, which could help to strengthenvaginal tissue. The presence of vitamin A increases the expression ofestrogen receptor alpha, vascular endothelial growth factor and the P2Y₂receptor gene in cervical epithelial cells. Such increases in expressionmay help to treat the symptoms of atrophic vaginitis.

Example 3 Vitamin A Increases Mucin-4 Expression Materials and Methods

RNA was isolated (as described above) from ME-180 cervical cells treatedwith 0 nM or 100 nm vitamin A in the absence of serum. The RNA wasreverse transcribed to form cDNA, and PCR techniques (as describedabove) were used to determine the effect of vitamin A on the expressionof mucin-4 (MUC-4).

Results

As shown in FIG. 6, addition of control β-actin primers leads tosynthesis of a 300 bp DNA product that is present in approximately equalamounts in all samples. This result indicates that approximately thesame amounts of template RNA were present in all samples. However, as isalso illustrated in FIG. 6, the MUC-4 product (800 bp) is found in thecervical cDNA control (lane 2). In ME-180 cells without vitamin A (FIG.6, lane 3), the MUC-4 product is absent. However, upon addition of 100nM vitamin A to ME-180 cells, MUC-4 expression is detectable after 4hours or 8 hours of treatment (FIG. 6, lanes 4 and 5, respectively).After 16 hours of treatment with 100 nM vitamin A (FIG. 6, lane 6),MUC-4 expression decreases but is still detectable. These data indicatethat low concentrations of vitamin A may be useful in the treatment ofatrophic vaginitis by increasing the expression of MUC-4.

Example 4 Stimulation of Keratinocyte Growth

This Example provides data showing the cell proliferating effect ofjasmonic acid and gibberellic acid on human skin keratinocytes.

Materials and Methods

A human skin keratinocyte cell line from Clonetics (Walkersville, Md.,normal human epidermal keratinocytes, neonatal, catalog number cc-2503)was exposed to the jasmonic acid and gibberellic acid (from SigmaChemical Co.) to determine their effect on proliferation ofkeratinocytes. The gibberellic acid used had the following structure(Gibberellin A₃).

The proliferative response of these human skin keratinocytes to the testcompounds was measured in a 96-well assay system using keratinocytebasal medium (KBM, Clonetics, catalog number CC-3103) as a control. Allcompounds were tested at three concentrations, 1×10⁻⁴ M, 1×10⁻⁵ M and1×10⁶ M. Cells were seeded into a 96 well plate at a concentration of2×10³ cells in 100 μl of KBM. The plate was incubated for 24 hours at37° C. in a humidified 5% CO₂ atmosphere. After incubation, 100 μl ofthe 1×10⁻⁴ M, 1×10⁻⁵ M or 1×10⁻⁶ M solutions of jasmonic acid andgibberellic acid were added to 5 wells each. In addition, 100 μl ofvehicle KBM was added to 5 wells as control. The plate was incubated for48 hours at 37° C. in a humidified, 5% CO₂ atmosphere. After incubation,20 μl of Cell Titer 96 Aqueous One Solution (Promega, Madison, Wis.) wasadded to all wells. The plate was swirled gently and placed back in theincubator for 3 hours. The spectrophotometric absorbance of each wellwas read at 490 nm.

Statistical analyses of data were performed using one-way ANOVA. P<0.05is considered significant, while P<0.0001 is considered extremelysignificant and P<0.001 is considered as very, very significant.

Results

Table 1 illustrates the cell proliferating effect of jasmonic acid onhuman keratinocytes, where the concentration of jasmonic acid variedbetween 1×10⁻⁴ M (designated JA4), 1×10⁻⁵ M (designated JA5) and 1×10⁻⁶M (designated JA6).

TABLE 1 Effect of Jasmonic Acid on Cell Proliferation Number Standard ofStandard Error of Group Points Mean Deviation Mean Median JA4 5 0.57320.02720 0.01216 0.5860 JA5 5 0.7628 0.03805 0.01702 0.7750 JA6 5 0.77340.06328 0.02830 0.7970 Control 5 0.5094 0.07334 0.03280 0.5050The data provided in Table 1 indicates that jasmonic acid has strongcell proliferating activity especially at the lower concentrations(P<0.001 at concentrations of 10⁻⁵ and 10⁻⁶ M). The effect on cellproliferation was greater at lower, rather than higher, concentrations.

Table 2 illustrates the cell proliferating effect of gibberellic acid onhuman keratinocytes, where the concentration of gibberellic acid variedbetween 1×10⁻⁴ M (designated GA4), 1×10⁻⁵ M (designated GA5) and 1×10⁻⁶M (designated GA6).

TABLE 2 Effect of Gibberellic acid on Cell Proliferation Number Standardof Standard Error of Group Points Mean Deviation Mean Median GA4 50.6512 0.01256 0.005616 0.6510 GA5 5 0.8184 0.04813 0.02152 0.8210 GA6 50.7854 0.04743 0.02121 0.7900 Control 5 0.5308 0.06833 0.03056 0.5220The data provided in Table 2 indicates that gibberellic acid has strongcell proliferating activity at all concentrations (P<0.001 at allconcentrations). The effect on cell proliferation was greater at lower,rather than higher, concentrations.

Example 5 Stimulation of Collagen Production

This example provides data showing that jasmonic acid and gibberellicacid stimulate collagen production.

The stimulation response of jasmonic acid and gibberellic acid(Gibberellin A₃, formula V) on collagen production in the human skinfibroblast cell line (Clonetics, Walkersville, Md., normal human dermalfibroblasts, neonatal, catalog number CC-2509) was measured using TakaraBiomedicals EIA assay kit (TAK MK101) sold by Panvera (Madison, Wis.).The cells were first grown in a 96-well assay system using Dulbecco'sModified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) bothpurchased from Sigma Chemical Co, St. Louis, Mo. Serum-free DMEM wasused as a control. All compounds were tested at three concentrations1×10⁻⁴ M, 1×0-5 M and 1×10⁻⁶ M. Cells were seeded into a 96 well plateat a concentration of 5×10³ cells in 100 μl of DMEM containing 10% fetalbovine serum (FBS, Sigma Chemical Co., St. Louis, Mo.). Plate wasincubated for 24 hours at 37° C. in a humidified 5% CO₂ atmosphere.After incubation, the medium was aspirated and the wells were rinsedtwice with 100 μl of serum-free DMEM. The final rinse was aspirated and100 μl of the 1×10⁻⁴ M, 1×10⁻⁵ M or 1×10⁻⁶ M solutions of the testcompounds were added to the wells (n=2 for each concentration). Inaddition, 100 μl of vehicle (serum-free DMEM) was added to 4 wells ascontrol. The plate was incubated for 48 hours at 37° C. in a humidified,5% CO₂ atmosphere.

The assay was done by using the recommended 20 ul of the supernatantfrom each well of the 96-well plate. Standard buffer and stop solutionswere freshly prepared before running the assay. 100 ul of antibody-PODconjugate solution (supplied with the kit) was added into the wellsusing pre antibody coated 96 well plate (supplied with the kit). 20 ulof standard and test solutions (from the other 96-well plate containingfibroblasts) were added to appropriate wells. Plate was mixed gently,sealed and incubated for three hrs. at 37° C.

After incubation each well was washed carefully four times with PBSbuffer (400 ul). All the wells were completely emptied at the end ofwashing from any liquid.

100 ul of substrate solution (hydrogen peroxide and tetramethylbenzidinein a buffer solution, supplied with the kit) was added to each well andthe plate was incubated for 15 minutes. At this point 100 ul of stopsolution (freshly prepared 1N H₂SO₄) was added to each well in the sameorder as substrate. The plate was gently mixed and absorbance was readat 450 nm.

Statistical analyses of data were performed using one-way ANOVA. P<0.05is considered significant, while P<0.0001 is considered extremelysignificant and P<0.001 is considered as very, very significant.

Results

Table 3 illustrates the collagen production of human fibroblast cellsexposed to varying concentrations of jasmonic acid and gibberellic acid.The concentration of jasmonic acid varied between 1×10⁻⁴ M (designatedJA4), 1×10⁻⁵ M (designated JA5) and 1×10⁻⁶ M (designated JA6). Theconcentration of gibberellic acid varied between 1×10⁻⁴ M (designatedGA4), 1×10⁻⁵ M (designated GA5) and 1×10⁻⁶ M (designated GA6).

TABLE 3 Effect of Jasmonic Acid or Gibberellic Acid on CollagenProduction by Human Fibroblasts Number Standard of Standard Error ofGroup Points Mean Deviation Mean Median JA4 2 0.8040 0.009899 0.0070000.8040 JA5 2 0.7895 0.006364 0.004500 0.7895 JA6 2 0.7475 0.0077780.005500 0.7475 GA4 2 0.7780 0.01838 0.01300 0.7780 GA5 2 0.8365 0.041720.02950 0.8365 GA6 2 0.8630 0.008485 0.006000 0.8630 CNA5 4 0.74830.006238 0.003119 0.7495 Control 4 0.7388 0.01431 0.007157 0.7330The data provided in Table 3 indicate that both jasmonic acid andgibberellic acid can stimulate collagen production in human fibroblasts.The effect of gibberellic acid on collagen production was more profound,particularly at lower, rather than higher, concentrations. However,jasmonic acid strongly stimulated collagen production at higherconcentrations.

Example 6 Stimulation of Fibronectin Production

This example provides data showing the effect of jasmonic acid andgibberellic acid on fibronectin production.

Materials and Methods

The stimulation response of jasmonic acid and gibberellic acid(Gibberellin A₃, formula V) on fibronectin production in the human skinfibroblast cell line (Clonetics, Walkersville, Md., normal human dermalfibroblasts, neonatal, catalog number CC-2509) was measured using TakaraBiomedicals EIA assay kit (TAK MK115) sold by Panvera (Madison, Wis.).The cells were first grown in a 96-well assay system using Dulbecco'sModified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) bothpurchased from Sigma Chemical Co, St. Louis, Mo. Serum-free DMEM wasused as a control. The compounds were tested in three concentrations1×10⁻⁴ M, 1×10⁻⁵ M and 1×10⁻⁶ M in duplicate. Cells were seeded into a96 well plate at a concentration of 1×10⁴ cells in 100 μl of DMEMcontaining 10% fetal bovine serum (FBS, Sigma Chemical Co., St. Louis,Mo.). The plate was incubated for 48 hours at 37° C. in a humidified 5%CO₂ atmosphere. After incubation, the medium was aspirated and the wellswere rinsed twice with 100 μl of serum-free DMEM. The final rinse wasaspirated and 100 μl of the 1×10⁻⁴ M, 1×10⁻⁵ M or 1×10⁻⁶ M solutions ofthe test compounds were added along with 100 μl of serum free DMEM tothe wells (n=2 for each concentration). In addition, 100 μl of vehicle(serum-free DMEM) was added to 2 wells as control. The plate wasincubated for 48 hours at 37° C. in a humidified, 5% CO₂ atmosphere.

The fibronectin assay was performed using the recommended 100 μl of thesupernatant from each well of the 96-well plate. Standard buffer andstop solutions were freshly prepared before running the assay. Apre-antibody-coated 96 well plate (provided with the kit) was used totransfer the test samples and control. The plate was mixed, sealed andincubated for 1 hour at 37° C. All the wells were washed after removingsample solutions, 3 times (300 μl) with washing buffer. 100 ul ofantibody-POD conjugate solution was added into the wells. Plate wasmixed, sealed and incubated for one hour at 37° C. Solutions wereremoved and the wells were washed 3 times with washing buffer. All thewells were completely emptied at the end of washing from any liquid. 100μl of substrate solution (hydrogen peroxide and tetramethylbenzidine ina buffered solution) was added to each well and the plate was incubatedat room temperature for 15 minutes. 100 μl of stop solution (freshlyprepared 1N H₂SO₄) was added to each well in the same order assubstrate. The plate was gently mixed and absorbance was read at 450 nm.

Statistical analyses of data were performed using one-way ANOVA. P<0.05is considered significant, while P<0.0001 is considered extremelysignificant and P<0.001 is considered as very, very significant.

Results

Table 4 illustrates the fibronectin production of human fibroblast cellsexposed to varying concentrations of jasmonic acid or gibberellic acid.

TABLE 4 Summary of Data Number Standard of Standard Error of GroupPoints Mean Deviation Mean Median JA4 2 0.2060 0.002828 0.002000 0.2060JA5 2 0.2025 0.0007071 0.0005000 0.2025 JA6 2 0.1955 0.0007071 0.00050000.1955 GA4 2 0.2150 0.005657 0.004000 0.2150 GA5 2 0.2090 0.0042430.003000 0.2090 GA6 2 0.2225 0.002121 0.001500 0.2225 Control 2 0.16600.004243 0.003000 0.1660

The concentration of jasmonic acid varied between 1×10⁻⁴ M (designatedJA4), 1×10⁻⁵ M (designated JA5) and 1×10⁻⁶ M (designated JA6). Theconcentration of gibberellic acid varied between 1×10⁻⁴ M (designatedGA4), 1×10⁻⁵ M (designated GA5) and 1×10⁻⁶ M (designated GA6).

Statistically all three compounds stimulated fibronectin production atall concentrations tested when compared with control. Hence, jasmonicacid and gibberellic acid are both effective for stimulating fibronectinproduction in human fibroblasts.

Example 7 Enhanced Human Skin Renewal

The example provides data showing that the rate of skin renewal isincreased by compositions containing jasmonic acid or gibberellic acidin healthy female volunteers.

The stratum corneum renewal time is the time taken for the wholethickness of the stratum corneum to be exfoliated and replaced by newcells from the dividing epidermis. This renewal time is a fundamentalindicator of skin health. Previous work has demonstrated that theturnover time of the stratum corneum can be measured non-intrusively byimpregnating the skin with a fluorescent marker dye that binds avidly tothe nonviable epidermal cells. See Jansen L H, Hojyo-Tomoko M T, KligmanA M., Improved fluorescence staining technique for estimating turnoverof the human stratum corneum, Br J Dermatol., 90, 9-12, 1974; Ridge B D,Batt M D, Palmer H E, Jarrett A, The dansyl chloride technique forstratum corneum renewal as an indicator of changes in epidermal mitoticactivity following topical treatment., Br J Dermatol., 118, 167-74,1988; Grove G L, Kligman A M, Age-associated changes in human epidermalcell renewal., J Geron., 38, 137-142, 1983. The time required for thedye to disappear is therefore an indication of the turnover time of thestratum corneum. Therefore, any differences in the time required for thedye to disappear from treated and non-treated sites can be considered tobe an expression of that product's ability to enhance epidermal renewal.

Materials and Methods

A dansyl chloride cell renewal study was conducted in healthy femalevolunteers ranging from 43-64 years of age. The study consisted of atwo-week treatment period during which time the products were appliedtopically twice daily, including on weekend days, to the test sites. Thearea of application was designated, approximately 5 cm×10 cm on theupper inner arm. The other arm served as a control with no productapplication. Two compounds, jasmonic acid (JA) and gibberellic acid (GA,Gibberellin A₃, formula V) were separately tested at a concentration of0.25% in hyaluronic acid (0.5% aqueous solution). Eight females receivedjasmonic acid and seven females received gibberellic acid.

The time required for a marker dye to disappear was used as the measureof cell renewal rate. These data are presented in Table 5.

TABLE 5 Time for Cell Renewal for Skin Treated with Jasmonic Acid (JA),Gibberellic Acid (GA) or Control JA CON- JA GA CON- GA PATIENT TROLRENEWAL PATIENT TROL RENEWAL # (Days) (Days) # (Days) (Days) 1 27 12 120 14 2 26 12 2 26 21 3 17 16 3 19 14 4 20 15 4 15 12 5 20 17 5 19 19 617 14 6 20 17 7 17 15 7 14 12 8 14 11As indicated by the data in Table 5, the time required for skin cellturnover is significantly reduced when the skin is treated withcompositions of either 0.25% jasmonic acid (JA) or 0.25% gibberellicacid (GA) compared to control skin that received no treatment. Jasmonicacid had an overall 28% faster cell renewal rate over control andgibberellic acid was 20% faster relative to control (FIG. 7). There wasno visible sign of any skin irritation or redness in any of the studyvolunteers.

Compounds known as alpha hydroxy acids (e.g. glycolic acid) have beenreported to decrease the cell renewal time. Moreover, treatment withalpha hydroxy acids is often accompanied by unwanted side effects suchas skin irritation and redness. The compositions of the invention maysatisfy an ongoing need for skin cell renewal anti-aging compositionsthat do not cause unwanted side effects and that are gentle to the skin.

Example 8 Jasmonic Acid Increases Epithelial Cell Growth

This Example illustrates that jasmonic acid can increase cellproliferation in a cervical epithelial cells.

Materials and Methods

Human cervical epithelial cells (ME-180, ATCC) were propagated inculture at 37° C. using 5% CO₂ in McCoy's 5A media (Invitrogen) with 10%heat-inactivated fetal bovine serum (Invitrogen). Jasmonic acid (Sigma)was diluted in the ME-180 media to 100, 10, and 1 microgram/mL. Athigher concentrations, the jasmonic acid turned the media yellow incolor, indicating that the pH was too low to accomidate normal cellgrowth.

ME-180 cells were plated in 12-well plates at a concentration of 5×10⁴cells per well in 2 mL of media. The cells were allowed to grow for 1day. Cells in three wells were counted by trypsinizing and using ahemocytometer to determine cell number on day zero. The media wasremoved from all other wells and was replaced with fresh media, 100, 10,or 1 μg/mL jasmonic acid-containing media (2 mL) in triplicate. Cellswere counted on days 1 and 2 to determine the effect of the jasmonicacid on cell growth.

Results

As shown in FIGS. 8 and 9, jasmonic acid increased epithelial cellgrowth after two days of incubation. Therefore, jasmonic acid, as wellas other plant hormones, may be useful in the treatment of atrophicvaginitis.

REFERENCES

These references and the other references cited herein are incorporatedby reference in their entirety.

-   U.S. Pat. No. 6,264,975 entitled, “Methods of hydrating mucosal    surfaces.”-   U.S. Pat. Nos. 5,981,506; 5,972,904; 5,958,897; 5,789,391 entitled,    “Method of treating sinusitis with UTPs and other related    compounds.”-   U.S. Pat. No. 6,277,855 entitled, “Method of treating dry eye    disease with nicotinic acetylcholine receptor agonists.”-   U.S. Pat. No. 6,200,981 entitled, “Pyrimidine derivatives.”-   U.S. Pat. No. 6,107,091 entitled, “Antisense inhibition of    G-alpha-16-expression.”-   U.S. Pat. No. 5,837,861 entitled, “Dinucleotides and their use as    modulators of mucociliary clearance and ciliary beat frequency.”-   U.S. Pat. No. 5,985,849 entitled, “Phosphate compounds and their use    as medicaments.”-   U.S. Pat. No. 6,107,297 entitled, “2,4-Dithi(oxo)-pyrimidin-5-yl    compounds bearing a tricyclic substituent useful as P2 purinoceptor    antagonists.”-   Garrad, R. C., Otero, M. A., Erb, L., Theiss, P. M., Clarke, L. L.,    Gonzalez, F. A., Turner, J. T., Weisman, G. A. “Structural basis of    agonist-induced desensitization and sequestration of the P2Y₂    nucleotide receptor,” Journal of Biological Chemistry 1998, 273(45),    29437-29444-   Gorodeski, G. I., Burfiend, P., Gan, S. U., Pal, D.,    Abdul-Karim, F. W. “Regulation by retinoids of P2Y₂ nucleotide    receptor mRNA in human uterine cervical cells,” American Journal of    Physiology 1998, C758-C765

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “a host cell” includes a plurality (forexample, a culture or population) of such host cells, and so forth.Under no circumstances may the patent be interpreted to be limited tothe specific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1-20. (canceled)
 21. A composition comprising an effective amount of avitamin E compound and a jasmonic acid compound and a pharmaceuticallyacceptable excipient for use in a vaginal cavity of a female mammal. 22.The composition of claim 21, wherein the composition increases growth ofepithelial or epidermal cells, increases expression of mucin-4, orpromotes faster cell turnover in the vaginal cavity.
 23. The compositionof claim 21, wherein the composition can increase expression of mucin-1,mucin-2, mucin-3A, mucin-3B, mucin-4, mucin-5B, mucin-5AC, mucin-6,mucin-7, mucin-11, mucin-13, mucin-15, mucin-17, mucin-19, or mucin-20genes.
 24. The composition of claim 21, wherein the vitamin E compoundis a compound of formula I or formula II:

wherein R1, R2, and R3 are independently hydrogen (H), methyl (CH₃) orhydroxyl (OH).
 25. The composition of claim 21, wherein the jasmonicacid compound is a compound of formula VII, VIII, IX, or X,

wherein: R4 is alkyl; R5 is COOR, or —(CH₂)_(n)—OX, where n is aninteger of from 1 to 20; R is H, or alkyl; X is H, or 1 to 6 hexose orpentose sugar residues; and Y is H, alkyl, or 1 to 6 hexose or pentosesugar residues.
 26. The composition of claim 21, wherein the compositionfurther comprises a retinoid or carotinoid.
 27. The composition of claim26, wherein the retinoid or carotenoid is vitamin A, beta-carotene,retinal, retinoic acid, or retinol.
 28. The composition of claim 26,wherein the composition increases expression of estrogen receptor alpha(ER-α) vascular endothelial growth factor, or P2Y₂ receptor.
 29. Thecomposition of claim 21, wherein the composition further comprises anucleoside or a nucleotide.
 30. The composition of claim 29, wherein thenucleotide is ATP.
 31. The composition of claim 21, wherein thecomposition is in the form of a lotion, cream, a gel, a foam, a spray oran aerosol.
 32. The composition of claim 21, wherein the composition isin the form of a dispersion or microemulsion.
 33. The composition ofclaim 21, wherein the composition is a component of a vaginal insert.34. The composition of claim 21, wherein the composition is a componentof a suppository.
 35. The composition of claim 21, wherein thecomposition further comprises a gibberellic acid compound.
 36. Thecomposition of claim 35, wherein the gibberellic acid compound is acompound of formula VI, VIa, or VIb: